Handbook of seed technology for genebanks …...Volume I of Handbook of Seed Technology for...

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CHAPTER 16. INTRODUCTION TO VOLUME II: THE ORGANIZATION OF CHAPTERS AND AN EXPLANATION OFABBREVIATIONS

CHAPTER 17. GENERAL APPROACHES TO PROMOTING SEED GERMINATION

CHAPTER 18. ACTINIDIACEAE

CHAPTER 19. AGAVACEAE

CHAPTER 20. AMARANTHACEAE

CHAPTER 21. ANACARDIACEAE

CHAPTER 22. ANNONACEAE

CHAPTER 23. AQUIFOLIACEAE

CHAPTER 24. ARACEAE

CHAPTER 25. BIGNONIACEAE

CHAPTER 26. BIXACEAE

CHAPTER 27. BROMELIACEAE

CHAPTER 28. CARICACEAE

CHAPTER 29. CHENOPODIACEAE

CHAPTER 30. COMPOSITAE

CHAPTER 31. CONVOLVULACEAE

CHAPTER 32. CRUCIFERAE

CHAPTER 33. CUCURBITACEAE

CHAPTER 34. DIOSCOREACEAE

CHAPTER 35. EBENACEAE

CHAPTER 36. ERICACEAE

CHAPTER 37. EUPHORBIACEAE

CHAPTER 38. FAGACEAE

CHAPTER 39. GRAMINEAE

CHAPTER 40. JUGLANDACEAE

CHAPTER 41. LABIATAE

CHAPTER 42. LECYTHIDACEAE

CHAPTER 43. LEGUMINOSAE

CHAPTER 44. LILIACEAE

CHAPTER 45. LINACEAE

CHAPTER 46. MALVACEAE

CHAPTER 47. MENISPERMACEAE

CHAPTER 48. MORACEAE

CHAPTER 49. MUSACEAE

CHAPTER 50. MYRTACEAE

CHAPTER 51. OLEACEAE

CHAPTER 52. OXALIDACEAE

CHAPTER 53. PALMACEAE

CHAPTER 54. PAPAVERACEAE

CHAPTER 55. PASSIFLORACEAE

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CHAPTER 56. PEDALIACEAE

CHAPTER 57. PIPERACEAE

CHAPTER 58. POLYGONACEAE

CHAPTER 59. PORTULACACEAE

CHAPTER 60. PROTEACEAE

CHAPTER 61. PUNICACEAE

CHAPTER 62. ROSACEAE

CHAPTER 63. RUBIACEAE

CHAPTER 64. RUTACEAE

CHAPTER 65. SAPINDACEAE

CHAPTER 66. SAXIFRAGACEAE

CHAPTER 67. SOLANACEAE

CHAPTER 68. STERCULIACEAE

CHAPTER 69. THEACEAE

CHAPTER 70. TILIACEAE

CHAPTER 71. TYPHACEAE

CHAPTER 72. UMBELLIFERAE

CHAPTER 73. URTICACEAE

CHAPTER 74. VITACEAE

CHAPTER 75. ZINGIBERACEAE

CHAPTER 76. GERMINATION TEST ENVIRONMENTS AND DORMANCY-BREAKING TREATMENTS FOR SPECIES IN OTHERFAMILIES

CHAPTER 16. INTRODUCTION TO VOLUME II: THE ORGANIZATION OF CHAPTERS AND AN EXPLANATION OF ABBREVIATIONS

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CHAPTER 16. INTRODUCTION TO VOLUME II: THEORGANIZATION OF CHAPTERS AND AN

EXPLANATION OF ABBREVIATIONSVolume I of Handbook of Seed Technology for Genebanks dealt with many of the principles ofseed testing which need to be understood when monitoring the viability of seed accessionsmaintained in gene banks. It will have become clear that one of the main problems facingthose who have the responsibility for monitoring seed viability in gene banks is that seeddormancy can often interfere with the results of germination tests designed to estimate thepercentage viability of accessions. The extent of the problem varies between species andbetween accessions within species, and the techniques which are most appropriate forminimising dormancy in germination tests also vary. In some species the problems aresufficiently understood so that prescriptions for germination tests have been developed whichenable dormancy to be removed completely. In other species sufficient is known to minimisethe problem of dormancy so that it is no longer a serious problem. However, there are stillmany species where existing techniques for dormancy removal are unsatisfactory, and yetothers where the information on dormancy is meagre.

This volume provides general approaches, detailed information, guidance and, whereavailable, prescriptions for removing dormancy and germinating the seeds. Since completelysatisfactory prescriptions are relatively rare, Chapter 17 deals with general approaches whichmay help staff in gene banks develop their own techniques for solving dormancy problems.

The subsequent chapters (Chapters 18 to 75) provide information, family by family, on thegermination of individual species of crop plants and sometimes their wild relatives. Thesechapters are essentially for consultation and, since the amount of information is large,considerable use is made of abbreviations. The final chapter (Chapter 76) summarises thegermination test recommendations which are available for species outside the 58 familiescovered by Chapters 18 to 75.

The rest of this chapter is essential to understanding Volume II since it explains the structureand abbreviations used. It also provides guidance on the preparation of solutions commonlyused in dormancy-breaking treatments.

THE STRUCTURE OF CHAPTERS 18 TO 75 AND ABBREVIATIONS USED INTHIS VOLUME

Each chapter which deals with a single family begins with a short introduction which includes,where available, the algorithm for devising dormancy-breaking techniques developed by staffat the Wakehurst Place Gene Bank (see Chapter 17). A comment is also provided on seedmorphology if this is considered to be of help in devising appropriate treatments to promotegermination. Prescriptions for germination test procedures and recommendations fordormancy-breaking treatments from various sources, but primarily the ISTA and AOSA rules,are tabled for species within genera where more detailed information is not provided within thechapter. Most alternating temperature regimes are diurnal cycles of 16h/8h and, to savespace, only exceptions to this general rule are noted in these tables. The followingabbreviations are used within these tables, and also Chapter 76, to describe the source ofinformation.

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AOSA AOSA (1981). Rules for testing seeds. Journal of Seed Technology, 6, 1-126.Atwater Atwater, B.R. (1980). Germination, dormancy and morphology of the seeds of herbaceous

ornamental plants. Seed Science and Technology, 8, 523-573.Ballard Ballard, L.A.T. (1972). High sensitivity to temperature of the germination responses of seeds of

Townsville stylo (Stylosanthes humilis H.B.K.). Proceedings of the International Seed TestingAssociation, 37, 779-791.

B&B Ballard, L.A.T. and Buchwald, T. (1971). A viability test for seeds of Townsville stylo usingthiourea, Australian Journal of Experimental Agriculture and Animal Husbandry, 11, 207-210.

Butler Butler, J.E. (1975). Germination of Stylosanthes humilis (Townsville stylo) in short cycles ofalternating temperature. Seed Science and Technology, 3, 523-528.

Cameron Cameron, D.F. (1967). Hardseededness and seed dormancy of Townsville lucerne (Stylosantheshumilis) selections. Australian Journal of Experimental Agriculture and Animal Husbandry, 7,237-240.

CHML Chin, H.F., Hor, Y.L. and Mohd Lassim, M.B. (1984). Identification of recalcitrant seeds. SeedScience and Technology, 12, 429-436.

Everson Everson, L. (1949). Preliminary studies to establish laboratory methods for the germination ofweed seed. Proceedings of the Association of Official Seed Analysts, 39, 84-89.

Fornerod Fornerod, C. (1975). Remarques sur la germination des semences potageres en laboratoire.Revue Horticole Suisse, 48, 6-9.

G&R Gordon, A.G. and Rowe, D.C.F. (1982). Seed Manual for Ornamental Trees and Shrubs.Forestry Commission Bulletin 59, 132pp., HMSO, London.

Heit Heit, C.E. (1948). Laboratory germination test results with herb and drug seed. Proceedings ofthe Association of Official Seed Analysts, 38, 58-62.

Holm Holm, A. McR. (1973). Laboratory procedures for germinating Townsville stylo seed pods.Journal of the Australian Institute of Agricultural Science, 39, 75-76.

ISTA ISTA (1985). International rules for seed testing. Seed Science and Technology, in press. (Weare most grateful to Dr. S. Cooper for providing draft copies of the new rules prior topublication.)

M&O Maguire, J.D. and Overland, A. (1959). Laboratory germination of seeds of weedy and nativeplants. Washington Agricultural Experiment Station Circular 349, 15pp.

McIvor McIvor, J.G. (1976). Germination characteristics of seven stylosanthes species. AustralianJournal of Experimental Agriculture and Animal Husbandry, 16, 723-728.

M&M Mott, J.J. and McKeon, G.M. (1979). Effect of heat treatments in breaking hardseededness infour species of Stylosanthes. Seed Science and Technology, 7, 15-25.

Oakes Oakes, A.J. (1984). Scarification and germination of seeds of Leucaena leucocephala (Lam.) DeWit. Tropical Agriculture (Trinidad), 61, 125-127.

O&W Olvera, E. and West, S.H. (1985). Aspects of germination of leucaena. Tropical Agriculture(Trinidad), 62, 68-72.

Riley Riley, J.M. (1981). Growing rare fruit from seed. California Rare Fruit Growers Yearbook, 13, 1-47.

R&S Rogers, B.J. and Stearns, F.W. (1955). Preliminary studies on the germination of weed seeds.Proceedings of the North Central Weed Control Conference, 12, 7.

SGCF Steinbauer, G.P., Grigsby, B., Correa, L. and Frank, P. (1955). A study of methods for obtaininglaboratory germination of certain weed seeds. Proceedings of the Association of Official SeedAnalysts, 45, 48-51.

Most chapters then go on to provide a more detailed summary and analysis of seedgermination responses to treatments, genus by genus, for the more important species whereinformation is available. This information is restricted mainly to orthodox species, butinformation on a few recalcitrant species is provided where dormancy is known to be apotential problem. The layout of information within each genus is as follows.

At the beginning of each genus a catalogue is provided of those species for which someinformation is given. The catalogue includes botanical synonyms and common names. It


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should be noted that sometimes identical synonyms may be given for species within anothergenus. Where this occurs and information is provided for the second genus the reader shouldconsider the information provided for both genera. The information on dormancy-breakingtechniques and germination test regimes for each genus is divided into seven sections. Notethat although the term dormancy is used in the titles below information on factors other thandormancy per se (see definition in Chapter 5, Volume I), particularly hardseededness (Chapter4, Volume I), is also included.

I. Evidence of dormancy

This section simply provides evidence of whether dormancy can be a problem and attempts toplace it in context - often by giving details of how long after harvest 'post-harvest' dormancytypically remains a problem under ambient conditions. Differences in the degree of dormancybetween species within the genus are sometimes noted. Other problems may be noted in thissection. For example, it is sometimes necessary to draw attention to the classification of seedstorage behaviour (see Chapter 1, Volume I) where this has been in some doubt.

II. Germination regimes for non-dormant seeds

In the majority of cases this section provides details of the prescribed germination testconditions for species within the genera given by the ISTA and the AOSA - where these areavailable. The ISTA and AOSA rules are divided into three parts here.

The first information is the method (or methods) of providing the medium for the germinationtest. The abbreviations used and their meanings are:

TP test on top of paper, that is, place the seed on filter papers, blotting papers or paper towelsin a petri-dish or similar container.

BP test between paper, including rolled paper towels and pleated papers.

S test in (sterilized) sand.

TS test on top of (sterilized) sand.

Often more than one medium is suggested. In this case choose whichever is the more suitablefor your laboratory.

The information after the first colon gives the prescribed temperature regime for thegermination test. An alternating temperature regime is denoted by A°/B°C (xh/yh), where A°Cis provided for x hours per day and B°C provided for the remaining y hours per day; e.g.20°/30°C (16h/8h) means germinate in a continuous alternating temperature regime in whichthe seeds are subjected to 20°C for 16 hours followed by 30°C for 8 hours each day. Oftenalternative temperature regimes are provided. The alternative regimes are separated by semi-colons.

The final information provided (after the second colon) is the total germination test period indays, but of course seedlings may have to be removed and counted at more frequentintervals. Moreover, it is likely that in many cases gene banks will have to continuegermination tests beyond these periods.

The provision of both the ISTA and the AOSA prescriptions for germination test regimes(where these are available for a species) allows the reader to compare and contrast the twosets. In the main these are quite similar, but where they occur the differences of detail shouldbe noted.

In addition to the ISTA and AOSA rules this section also includes other published information,


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where available, on germination test procedures which are satisfactory for non-dormantseeds.

III. Unsuccessful dormancy-breaking treatments

The third section gives details of treatments that have been applied to seeds in attempts tobreak dormancy, but which have either failed to increase germination more than marginally ormay have even led to a reduction in germination - either by inducing dormancy in the seeds orby damaging the seeds in some way. Although at first sight the reader may consider thisinformation to be of no interest - after all the requirement is to promote germination - it isimportant to be aware of those treatments which should be avoided. Moreover, the reader willbegin to notice that similar treatments may appear in more than one section. These apparentinconsistencies and contradictions, often between different reports, are nevertheless probablyindicative of the real situation: a treatment which greatly promotes the germination of seeds ofone accession, may fail to promote the germination of seeds of another accession, whilst in athird accession germination may be reduced by the treatment. Hence the inclusion of thisinformation here.

IV. Partly-successful dormancy-breaking treatments

Treatments detailed in this section have promoted the germination of some dormant seeds,but have either failed to promote the germination of all the dormant seeds within a singleaccession, or within a report they may have promoted full germination in some accessions butnot promoted full germination in other accessions.

V. Successful dormancy-breaking treatments

The ISTA and/or the AOSA recommendations for breaking dormancy are provided first of all inthis section (where available for a species). The style of layout is different from, and lessdetailed than, that given for other sources of information. The reasons for this are to highlightthe ISTA and AOSA recommendations and to avoid repetition of the treatment details, whichare given below.

Pre-chill: Seeds are placed in contact with the moist substratum and kept at a low temperaturefor an initial period before being moved to the germination test temperature. With theexception of tree seeds, the pre-chill temperature is between 5° and 10°C and the initialtreatment period up to 7 days, although in some cases - particularly the more dormant of thegrasses - this may be extended to 14, or, rarely, 28 days. Tree seeds are kept at 3°-5°C forbetween 7 days and a year.

Pre-dry: Before imbibition the dry seeds are heated at a temperature not exceeding 40°C withfree air circulation for up to 7 days.

Potassium nitrate: The germination test paper is moistened with a 0.2% solution of potassiumnitrate (see below for details of solution preparation).

GA3: The germination test paper is moistened with 200-1000 ppm of gibberellic acid (seebelow for details of solution preparation).

Pre-wash: Seeds are soaked and washed in running water at between 20° to 25°C for 2 hoursor so to remove substances in seed (or fruit) coats which may inhibit germination.

Test at: An alternative germination test regime is suggested if difficulties are encountered atthe prescribed germination test temperatures (given in II. Germination regimes for non-dormant seeds).


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The information which follows the ISTA and AOSA recommendations (where available)provides details of those treatments which have been reported to be fully effective in promotingthe germination of all, or nearly all, dormant seeds within accessions. Note, however, thatthese treatments may on occasion be the same as those given in the preceding sections: thatis a treatment found to be successful for one seed lot may not have been successful whenapplied by another worker to a different seed lot.

VI. Comment

This section may point out problems with the ISTA/AOSA prescriptions and recommendations,conflicts between various reports in the literature and attempt to provide a guide to devisingappropriate germination test regimes. In a few cases germination test prescriptions may begiven; in rather more cases the more suitable techniques will be suggested with alternatives inthe event of failure. The symbol A in this section indicates unpublished work by the authors ofthis report.

VII. References

Within each genus the numbers in brackets refer to the references provided in the last section.These are numbered in alphabetical order with the exception of those references added infinal revisions of this manuscript. It is envisaged that gene bank staff will consult only a veryfew of these references, if at all. Most, if not all, of the relevant information has been extractedand summarised in the sections I to VI.

Shorthand used to describe treatments

A shorthand notation has been devised to present the information in as concise a form aspossible. A description of the treatment is given before the colon; the information following thecolon gives precise treatment details - where available. Some examples follow.

Alternating temperatures: (4); 20°/30°C, 20°/35°C (16h/8h) (8)

Potassium nitrate: pre-applied, 24h, 0.1-1% (7); co-applied, 0.1, 0.2%, at 25°C (6)

Light: (10); dark, continuous (12); red, 15 min/d (3)

These have the following meaning:

Reference 4 reported that alternating temperatures were used but no treatment details weregiven. Reference 8 applied alternating temperatures of either 20°C for 16 hours per day and30°C for 8 hours per day or 20°C for 16 hours per day and 35°C for 8 hours per day.Reference 7 treated the seeds to potassium nitrate solutions between 0.1 and 1% - withseveral intermediate concentrations - for one day before beginning the germination test whichwas then carried out on a substrate moistened with water - hence pre-applied. Reference 6moistened the germination test substratum - hence co-applied - with either 0.1% or 0.2%potassium nitrate (but at no intermediate concentrations) and the germination test was at aconstant temperature of 25°C. Reference 10 reported that a light treatment was given but nodetails were reported. Reference 12 carried out the germination test in the dark. Reference 3exposed the seeds to red light, but only for 15 minutes per day.

Often incomplete treatment details are provided. References 4 and 10 above provideexamples of the layout in such cases. Incompleteness is usually because the information wasnot provided by the paper referred to, but sometimes we have omitted information thatappears to us to be mistaken or misleading.

It is possible that mistakes in interpretation or transcription may have been made. Weapologise to the authors of any papers cited if this has occurred and would welcome


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correspondence pointing out any errors or omissions, and particularly welcome further detailsof successful dormancy-breaking treatments.

In passing it should be noted that certain regimes are referred to very frequently. For example,diurnal alternating temperature regimes of 20°/30°C, where the higher temperature is appliedfor 8 hours per day combined with co-application of 0.2% potassium nitrate are oftenmentioned. This is because it is a germination test regime recommended by ISTA/AOSA for alarge number of species and a large number of workers have tested the response of seedgermination to this regime as a consequence. However, often this regime appears superior bydefault - since other regimes will not necessarily have been tested. Consequently the reader isreminded that the information reported here is in that sense limited: other, more favourable,germination test regimes and dormancy-breaking treatments may exist which have not yetbeen the subject of investigation.

Abbreviations

The following abbreviations have been used to provide treatment details.

cm-2

per square centimeter

°C degrees Celsiusd dayg grammeGA gibberellins, the subscript denoting the particular gibberellin; GA3 is the most commonly applied

gibberellin.h hourj joulekc kilocycles, that is 1000 cyclesl litrem month

m-2 per square metre

M Molar, that is the molecular weight in grammes dissolved in a litremin minuteml millilitre, that is 10-3 litremol 6.02x1023 photons - see Chapter 6, Volume IN normal, that is the number of gramme-equivalents of the substance dissolved in a litre of solution

where one gramme equivalent equals the gramme-molecular weight of the substance divided by itshydrogen equivalence.

nm nanometre, that is 10-9 meteres - see Chapter 6, Volume IpH concentration of hydrogen ions given as the negative logarithm of hydrogen ion activityppm parts per million, equivalent to 0.0001% (see below)R Roentgen, a unit of ionizing radiations second

s-1 per second

W watt - see Chapter 6, Volume I/ between two temperatures or time period indicates an alternating regime (usually alternating

temperature)/ followed by another symbol indicates per, e.g./d = per day% percentage concentration, usually in terms of weight per volume (w/v), that is g/100 ml of solution

How to utilise the information provided for each genus


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It is not intended that all seven sections of the information on seed germination provided foreach genus should necessarily be read in sequence. The following approach is suggested.

After reading the introduction to the family - and Table 17.1 or Table 17.2 if either is referred to- read sections I (Evidence of dormancy) and VI (Comment).

In most cases these sections will provide sufficient information to decide upon a suitablegermination test procedure and whether to apply one or more dormancy-breaking treatments -and, if so, the details of these treatments. Reference to sections II (Germination regimesfor non-dormant seeds) and V (Successful dormancy-breaking treatments)may help to clarify the details of these treatments and procedures.

The information provided in sections III (Unsuccessful dormancy-breakingtreatments) and IV (Partly-successful dormancy-breaking treatments) shouldhelp the reader to understand why certain dormancy-breaking treatments and germination testprocedures are to be preferred and why others are best avoided. This information, however,will probably be of more use to those attempting to devise and develop improved germinationtest procedures and dormancy-breaking treatments if the advice presently available(Comment) is found to be inadequate or inappropriate for an accession.

For gene banks handling comparatively few genera Section VII (References) could formthe basis of a reference library of articles on seed germination and dormancy. However this isnot essential in view of the information summarised in this manual. More useful will be theinformation generated from the results of germination tests on material maintained within thegene bank.

Commencing an alternating temperature germination test

If seeds are to be tested in an alternating temperature regime it must be decided which of thetwo temperatures the seeds are exposed to first. There are three main possibilities:

(1) Initially expose the seeds to the first stated temperature of the alternatingtemperature regime. For example, in the case of the regime 20°/35°C (16h/8h) theseeds would be exposed to 20°C for 16 hours before their first exposure to 35°C.

(2) Expose the seeds to the lower of the two temperatures first. For example, inthe above alternating temperature regime 20°C is the lower temperature and theseeds are thus exposed to 20°C for 16 hours before their first exposure to 35°C.

(3) Expose the seeds to the longer duration of the two phases first. In the aboveexample 20°C is applied for the longer duration and thus the seeds are exposed to20°C for 16 hours before their first exposure to 35°C.

Where a regime is described as, for example, 30°/10°C (16h/8h) it will be seen that it is notpossible to satisfy all three possible rules. We suggest that it is logical for the first statedtemperature to be the initial temperature to which the seeds are exposed and that, therefore,the first rule should take priority over all others. It is important that the protocol to describealternating temperature regimes be explicitly stated and consistently applied in gene banks.

Application of light during part of alternating temperature cycles

When light is applied during part of an alternating temperature cycle it is the usual practice forthe light treatment to coincide with the higher temperature phase (as would occur in a naturalenvironment). If the light is applied once a day for a lengthy period, it is also convenient for theduration of any light treatment to be the same as that for the higher temperature phase of thealternating temperature cycle. This is because the heat generated by the lights (even


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fluorescent tubes) will affect the maintenance of temperature. Thus if the periods of exposureto the higher temperature and to light coincide throughout then the higher temperature of thegermination environment can be set taking into account the heat generated by the lights. It isimportant that the protocol adopted for the provision of light during alternating temperatureregimes be explicitly stated and adhered to. For more information on light and seedgermination see Chapter 6, Volume I.

MAKING UP SOLUTIONS

Two of the more common dormancy-breaking treatments pre-applied or co-applied to seedsare potassium nitrate and gibberellic acid. The preparation of solutions of these and othercompounds will be required in most, if not all, gene banks. Consequently some notes on thepreparation of solutions are provided below.

To make up a 0.2% solution of potassium nitrate, 2 g of potassium nitrate is dissolved in onelitre of distilled or deionised water. (It is not essential to make up a whole litre of solution. Forexample, 1 g dissolved in 500 ml would also provide a 0.2% solution.) To make up a 500 ppmsolution of GA3 dissolve 0.5 g GA3 in one litre of distilled or deionised water. GA3 generallytakes a long time to dissolve in water and considerable stirring; with a glass rod, may berequired before all the GA3 has dissolved. Strong concentrations of GA3, above 800 ppm, willreduce pH. To avoid this it is generally advised to use a buffer solution of 0.01 M di-sodiumhydrogen orthophosphate dihydrate/di-sodium hydrogen orthophosphate monohydrate for GA3concentrations of 800 ppm and above. This solution is prepared by dissolving 1.7799 g of di-sodium hydrogen orthophosphate di-hydrate (Na2HPO42H2O) and 1.3799 of di-sodiumhydrogen orthophosphate monohydrate (Na2HPO4H2O) in distilled or deionised water andmaking up to one litre. The GA3 is then dissolved in this buffer solution.

Solution concentrations

Throughout the report the concentrations of solutions of potential dormancy-breaking agentsare expressed in the style given by the reference. To enable the reader to convert betweenthese different forms of presentation, Figure 16.1 has been provided. In particular thegrammes per litre scale (g/1, or g 1-1) provides sufficient information to enable the reader tomake up the required concentrations of solutions. The only additional information required touse Figure 16.1 is the molecular weight of the potential dormancy-breaking agent. These areusually provided on the labels of chemical containers. Some values are provided here in Table16.1, but more comprehensive information is available from chemical company catalogues andMerck's Index. The latter is particularly recommended. The use of Figure 16.1 is described inthe caption.

ADDITIONS AND AMENDMENTS

The chapters which follow are very much a first attempt at pooling practical informationconcerning methods of overcoming seed dormancy and promoting germination. It is intendedthat this report should be referred to on a day to day basis. Readers might like to use thespace after the information on each genus to append their own notes of any additionalinformation they consider useful. Perhaps the most striking point illustrated by the format usedhere is how little is known of satisfactory treatments to break dormancy in seed of so manyspecies. Note that whilst the literature on seed dormancy is considerable, that concerned withregimes capable of promoting full germination for many accessions is extremely limited. Theauthors hope that this realisation will spur readers on to add to this knowledge. We welcomereports of successful dormancy-breaking treatments which can be included in subsequentrevisions or amendments to this handbook.


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TABLE 16.1. The molecular weights of selected compounds which have been applied toseeds as putative dormancy-breaking agents.

Molecular Weight

Abscisic acid 264

Acetaldehyde 44

Acetamide 59

Acetic acid 60

Alanine 89

Ammonium bisulphide 51

Ammonium bisulphite 99

Ammonium chloride 53

Ammonium nitrate 80

Ammonium phosphate, dibasic 132

Ammonium phosphate, monobasic 115

Ammonium sulphate 132

Ammonium sulphide 68

Ammonium sulphite 116

Ascorbic acid 176

Boric acid 62

Calcium hypochlorite 143

Calcium nitrate 164

Diethyl dithiocarbanate, sodium 171

Dimercaprol (Dithioglycerol) 124

Dinitrophenol 184

Dithiothreitol 154

Ethrel (Ethephon or CEPA) 144

Gibberellic acid 346

Hydrogen peroxide 34

Hydrogen sulphide 34

Hydroxylamine hydrochloride 70

Indoleacetic acid 186

Ketoglutaric acid 146

Kinetin 215

Mercaptoethanol 78

Methylene blue 374

Napthaleneacetic acid 186

Nitric acid 63

Potassium cyanide 65

Potassium nitrate 101

Potassium nitrite 85

Potassium permanganate 158

Potassium sulphate 174

Potassium thiocyanate 97

Potassium thiosulphate 190


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Sodium azide 65

Sodium fluoride 42

Sodium hydroxide 40

Sodium hypochlorite 74

Sodium nitrate 85

Sodium nitrite 69

Sodium sulphide 78

Sodium thiocarbonate 154

Sodium thiocyanate 81

Sodium thiosulphate 158

Sucrose 342

Sulphuric acid 98

Thiourea 76

Uranyl nitrate 394

Urea 60

How to use Figure 16.1

The first three scales of the nomograph differ by factors divisible by ten. To convert betweenthese values place a ruler on the diagram perpendicular to these axes and read off the values.To determine molarity connect up the concentration in grammes per litre with the molecularweight of the compound (see Table 16.1) with the straight edge of a ruler. The point where theruler crosses the molarity scale gives the value of the molarity of the solution. Alternatively if itwere required to make up a solution of a given molarity, connect up this value with themolecular weight of the compound with the straight edge of a ruler and note the point on thegrammes per litre scale where the straight edge crosses.

Two examples of the use of the nomograph are shown by broken lines. The uppermost brokenline is for potassium nitrate - molecular weight 101. A 0.2% solution is the same as a 2000ppm solution and is made by dissolving 2 grammes of potassium nitrate in one litre. Theresultant solution can also be described as 0.02 M or as 2x10-2 M. The lower broken dottedline is for GA3 - molecular weight 346. Thus, for example, a 500 ppm GA3 solution is 0.0015 M(1.5x10-3 M) and can be obtained by dissolving 0.5 grammes of GA3 in one litre of solution.


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CHAPTER 17. GENERAL APPROACHES TO PROMOTING SEEDGERMINATION

Deciding appropriate germination test regimes for accessions so that germination tests reflect true viability with minimuminterference from dormancy (and other factors which limit germination, such as hardseededness) can be one of the moredifficult decisions which gene bank staff need to make. In the following chapters we have summarised useful information onthe germination response of seeds of various genera to very many test regimes and dormancy-breaking treatments togetherwith advice on suggested test procedures. In many cases, however, the information available is far from complete andaccordingly much of the advice is very tentative. In such cases it is hoped that gene bank staff will be able to improve on thesuggestions made. In order to help in this endeavour, in this chapter we discuss four different types of approach todetermining appropriate germination environments.

ECOLOGICAL GUIDELINES TO DEVELOPING APPROPRIATE GERMINATION ENVIRONMENTS

A consideration of the ecology of a species may sometimes help to provide some guidance as to appropriate germination testconditions. At the simplest level tropical species generally require higher temperatures for germination than temperate species.However, there are other more subtle responses which relate to the strategy of the plant in relation to its natural environmentand a consideration of these may help in the development of germination test conditions which minimise dormancy.

Dormancy may prevent seeds from germinating in the wrong place at the wrong time

As mentioned in Chapter 5 (Volume I) the majority of mature seeds show innate (or inherent or primary) dormancy - acondition necessary to prevent germination on the mother plant before the seed is shed. Innate seed dormancy in mostspecies tends to be gradually lost with time (except in some tree species), often at a rate depending on temperature andmoisture content. But induced (or secondary) dormancy, in many respects similar to innate dormancy, may subsequently beinduced by conditions which signal that the current environment is inappropriate for germination - e.g. high temperatures oranaerobic conditions. Furthermore enforced dormancy may be temporarily imposed by conditions which again appear to play arole in ensuring that the seed only germinates when there is a reasonable probability of the seedling growing to maturity.Dormancy in all its forms (see Chapter 5, Volume I) therefore tends to prevent seeds from germinating in the wrong place atthe wrong time.

Field environment

For example, many annual or ephemeral species produce large numbers of small seeds. At any one time it is common forthere to be more dormant seeds in the soil waiting for appropriate conditions than there are growing plants. Since small seedsonly contain small food reserves, only a small amount of seedling growth can occur before the seedling needs to startphotosynthesising. Thus germination more than a few centimetres below the soil surface or beneath dense vegetation couldlead to seedling destruction. It would seem that many species have evolved dormancy mechanisms which avoid theseproblems. The seed needs to be able to respond to environmental factors that signal when it is at or near the soil surface.There seem to be two major environmental factors which many species use as indicators - light and alternating temperatures;and in most cases the seeds respond to both. Light is obviously only present at or near the soil surface; and diurnaltemperature alternations have a maximum amplitude (or diurnal range) at the soil surface: the range rapidly diminishes withdepth in the soil profile. Furthermore even at the soil surface the amplitude is diminished by the presence of vegetation. Sincevegetation contains chlorophyll which absorbs strongly in the red region of the spectrum, the quality of light beneath vegetationmay be inhibiting since far-red light penetrates much more and thus the light quality tends to affect the balance of phytochromein the seeds in favour of the inactive Pr form (see Chapter 6, Volume I).

Accordingly it is very common for small seeds of annuals and ephemerals to be light-sensitive and to germinate in response towhite (or red) light and alternating temperatures. In many cases neither of these factors on their own has a major effect andboth are necessary for a maximum response. Both these factors also often interact with nitrate ions.

Maternal environment

It may be worth considering the maternal environment in which the seeds develop as a further guide to distinguishing thosespecies likely to require light treatments in order to promote seed germination. The light-filtering properties of the maternaltissues which surround the developing seeds, whilst they remain moist, can affect the sensitivity of seed germination to light insubsequent environments. Where the maternal tissues have a high chlorophyll content (that is are green) the seeds tend torequire a light stimulus for germination (in subsequent tests). In contrast those seeds which have developed within maternaltissues where the chlorophyll content declined whilst the seeds remained moist tend to be able to germinate subsequently inlight or darkness (that is are light insensitive). This is because the seeds developing within green maternal tissues would havereceived light filtered by the chlorophyll. This light would have a low red/far red ratio and most of the phytochrome would be inthe inactive Pr form. In the latter case, however, the light would not have been filtered to such an extent; the red/far red ratio ofthe light received by the seeds would have been greater and thus more of the phytochrome would be in the active Pfr formwhich promotes germination. This Pfr would remain after desiccation and subsequent imbibition and accounts for theinsensitivity of these seeds to light.

Large-seeded species of arid environments

As mentioned in Chapter 6 (Volume I), sustained and/or high-intensity light can also inhibit the germination of some seeds.This response is more common amongst somewhat larger seeds of species of arid environments, where it is more important

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for seeds to germinate below the soil surface where short-term severe water shortage is less likely, and the probability of theshoot not reaching the soil surface before food reserves are reduced is lessened by the larger food reserves of the seed.

In desert regions where rains are infrequent and of limited duration, ephemeral plants have short life cycles and it is importantfor them to germinate only after a substantial fall of rain (and not after a minor shower which would not provide sufficient waterfor survival). In such cases seeds of some species appear to have their own 'rain gauge' which depends on the fact that ittakes a certain minimal amount of rain to leach germination inhibitors. Thus in such cases germination may be stimulated bywashing the seeds in running water.

Probability of plant establishment and survival

Dormancy mechanisms often also tend to ensure that the seeds do not germinate at times when the probability of the survivalof the plant to maturity is poor. For example, many seeds in temperate latitudes tend to germinate after the winter, i.e. at thebeginning of the main growing season. These seasonal considerations are often independent of seed size and thus manytemperate species germinate best after a period of stratification (i.e. cool temperatures applied to moist seeds). Such aresponse ensures that seeds do not germinate at the beginning of winter. In contrast, seeds of species from Mediterraneanclimates tend to avoid germination at the beginning of the hot, dry summer and germination at the beginning of the cool moistwinter is more common. In such cases it would be less likely to find marked responses to stratification. This would also begenerally true of tropical plants, but see the note below.

In tropical rain forests the temperature and water supply is always adequate for growth and so seeds of such species oftenshow little dormancy. Seeds of many of the woody perennial species are also recalcitrant. Such seeds do not have to survivelong periods in the dry conditions, and fresh supplies of short-lived seeds are produced regularly by the mature plants. Manyseeds of this type show little dormancy, and many are killed by cool temperatures (e.g. less than 10° to 15°C) which theywould never normally experience. However, even in tropical forests some species are not recalcitrant, and some may showlight responses to ensure that they only germinate when a gap arises in the forest canopy and/or the soil is disturbed afterclearance, thus ensuring that early growth is not inhibited by low light intensity.

There are, of course, exceptions to these general principles. In particular it should be noted that laboratory treatments whichemulate environments which the seeds would never experience in their natural habitat may sometimes promote germination.For example, low temperatures applied to moist seeds of some species of tropical and sub-tropical ecotypes can promotegermination. Nevertheless the few examples above may be sufficient to indicate that a consideration of the ecology of thespecies may give useful clues as to the type of laboratory germination techniques which may be most appropriate.

Further reading

Cresswell, E.G. and Grime, J.P. (1981). Induction of a light requirement during seed development and its ecologicalsignificance. Nature, 291, 583-585.

Grime, J.P., Mason, G., Curtis, A.V., Rodman, J., Band, S.R., Mowforth, M.A.G., Neal, A.M. and Shaw, S. (1981). Acomparative study of germination characteristics in a local flora. Journal of Ecology, 69, 1017-1059.

THE ANATOMICAL APPROACH TO DEVELOPING APPROPRIATE GERMINATION ENVIRONMENTS

This approach has been developed largely by B.R. Atwater from long experience of attempting to germinate seeds of flowerand ornamental species. Atwater's observations suggest that dormancy and germination requirements in these species areclosely related to the maturity of the embryo and the structure and permeability of the seed coat coverings in the mature seed.

Atwater has described eight basic forms of seed structure. These categories have been described in Chapter 3 (Volume I).Within each of these categories it is suggested that the seeds have similar dormancy and germination patterns, but it shouldbe emphasised that the mechanisms suggested are hypotheses rather than established facts.

I. Seeds with dominant endosperm (ENDOSPERMIC SEEDS) and immature dependent embryos (that isembryos not ready to germinate)

A. BASAL RUDIMENTARY EMBRYOS

The rudimentary embryos are the principal cause of delayed germination in this group. Inhibitors found in the endosperm mayalso contribute to the delay. Treatment consists of neutralization of the inhibitors with leaching, or with extra oxygen at lowtemperature followed by higher temperatures favourable to rapid embryo growth. Gibberellic acid increases the rate of embryodevelopment if added to the substrate.

B. AXILLARY LINEAR EMBRYOS

Linear embryos are similar to the above. An additional block is added by the seed coats which may limit oxygen entry, butseed coat permeability may be increased when the seeds are exposed to light. Gibberellic acid added to the substrateincreases the rate of embryo development.

C. AXILLARY MINIATURE EMBRYOS

Miniature seeds having minimal embryos are protected by thin seed coats showing a light requirement for oxygen permeability.Germination is prompt. Gibberellic acid is an aid, but not a substitute for light.

D. PERIPHERAL LINEAR EMBRYOS

Peripheral embryos which surround the food storage organ rather than being contained within it have multiple seed coveringswhich contain inhibitors, and which form barriers to both oxygen and water. Treatment is primarily leaching of the inhibitor but


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cold or light treatments may be required to increase permeability. Removal of the seed covering structures is also effective.

Summaries of seed anatomy, blocks to germination, successful germination test regimes and families exhibiting thesecharacteristics are provided in Table 17.1 for endospermic seeds.

TABLE 17.1 ENDOSPERMIC SEEDS: Anatomy and Germination

A. BASALRUDIMENTARY

EMBRYO

B. AXILLARYLINEAR EMBRYO

C. AXILLARY MINIATUREEMBRYO

D. PERIPHERAL LINEAR EMBRYO

SEED ANATOMY

embryo small, show littledifferentiation,

linear in a centralaxillary

linear-spatulate in acentral

curved in a peripheral position

basal location Position axillary position surrounding the perisperm orendosperm

cotyledons obscure and limitedto a few cells

minimal: thin, narrowand shorter than thestalk

not expanded and equal tothe stalk

thin, narrow and equal to the stalk

endosperm occupies most ofseed and surroundsembryo

occupies half ormore of seed andsurrounds thecentral embryo

occupies half or less of theseed and surrounds thecentral embryo

centrally placed within the curvedenvelope

seed coat permeable andfibrous orreticulated

thin, reticulous orfibrous: may besemi-permeable

thin and fragile thin testa, leathery outer coat andoften parts of the calyx

seed size medium, 2-4 mmlong

medium-large, 3-10mm long

small, 1 mm long or less medium-large, 2-6 mm diameter

example Anemone coranaria Cyclamen persicum Nemesia strumosa Portulaca grandiflora

embryo must developbefore germination,hence delay

must develop beforegermination, hencedelay

BLOCKS TOGERMINATION

endosperm inhibitors togermination may bepresent

seed coat permeable, noblock togermination

may be semi-permeable

permeable if light provided may be impermeable and containinhibitors

temperature temperate species15°C; others 20°C,20°/30°C

15° to 20°C;10°/30°C or20°/30°C

10°, 15° or 20°C;20°/30°C, 10°/20°C or5°/30°C

10°, 15° or 20°C (rarely 3°-6°C);20°/30°C

testduration

14-28d; in extremecases 100-155d


10-28d; in extreme cases40-60d

5-28d; in extreme cases 65-75d

KNO3 *0.2% *0.2% *0.2% 0.2%

SUCCESSFULGERMINATION

TEST

GA3 *100-400 ppm *400-800 ppm 120-400 ppm

pre-chill 2w at 3°-5°C, or(rarely) testthroughout at 5°-8°C

8w at 5°C 2-4w at 5°C

REGIMES(*most widely

applied treatments)

light probably insensitive *insensitive orsensitive (promotory,may aid imbibition)

*sensitive (promotory);supply red light for up to70h either continuously or12h per day

*may be insensitive or sensitive(promotory)

seed coats partial removal ofpericarp

may be necessary toremove endospermover radicle

*remove (calyx and) outer coats;pierce, chip (clip)

pre-soak hot water; 0.5h in1N KOH or otheralkaline solutions

pre-wash in water

PLANT FAMILIESWITH SPECIES

EXHIBITING THESECHARACTERISTICS

ARALIACEAE,FUMARIACEAE,PAPAVERACEAE,RANUNCULACEAE

ERICACEAE,GENTIANACEAE,PRIMULACEAE,RESEDACEAE,SOLANACEAE,TROPAEOLACEAE,UMBELLIFERAE

BEGONIACEAE,CAMPANULACEAE,CRASSULACEAE,HYPERICACEAE,LOBELIACEAE,SAXIFRAGACEAE,SCROPHULARIACEAE,SOLANACEAE

AIZOACEAE, AMARANTHACEAE,CACTACEAE, CAPPARIDACEAE,CARYOPHYLLACEAE,CHENOPODIACEAE,MESEMBRYANTHACEAE,NYCTAGINACEAE,POLYGONACEAE,PORTULACACEAE

TABLE 17.2 NON-ENDOSPERMIC SEEDS: Anatomy and Germination

A. AXILE FOLIAREMBRYOS WITH

HARD SEED COATS

B. AXILE FOLIAREMBRYOS WITH

THIN,MUCILAGINOUS,

SEED COATS

C. AXILE FOLIAR EMBRYOS WITHWOODY SEED COATS AND INNER SEMI-

PERMEABLE LAYER

D. AXILEFOLIAR

EMBRYOSWITH FIBROUS

SEED COATAND


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SEPARATEINNER SEMI-PERMEABLE

MEMBRANOUSLAYER

SEED ANATOMY

embryo spatulate, bent orfolded and fills seedcavity

spatulate or bentand fills most ofseed cavity

fills most of seed cavity spatulate andfills most ofseed cavity

cotyledons large, thickened anddominant over thestalk

large, thickened anddominant over thestalk

expanded and dominant large anddominantembryo

endosperm reduced to a thinlayer or lacking

thin layer aroundembryo or lacking

surrounds axillary embryo forming a lining ofthe seedcoat or none

thin layer(s)lining themembranoustesta

seed coat hard thin, may exudemucilage when wet,or contain amucilaginous layerwithin

woody, usually achenes with fused orseparate membranous testa

seed size medium-large; 2-15mm long

small-medium; 1-6mm long

medium-large; 2-10 mm long wedge-shapedachenes; 1-10mm long

example Ipomoea purpurea Iberis amara Verbena x hybrida Dimorphothecasinuata

embryo inhibitorspresent incotyledons

BLOCKS TOGERMINATION

endosperm possibility ofinhibitors in residualendosperm

impermeable tooxygen,gibberellins andinhibitors

seed coat impermeable whendesiccated; imbibitionthrough specializedvalves or afterscarification

becomesimpermeable tooxygen and othergases as it absorbswater

permeable to moisture, but impermeable tosome gases and inhibitors may be presentwithin the seed coat

inhibitors maybe present

temperature 20°C; 20°/30°C 15°C, 20°C;15°/25°C or10°/30°C

15°, 20°, 25°C; 20°/30°C, 25°/35°C,15°/25°C or 15°/30°C

15°,20°C;15°/25°C or20°/30°C

testduration



9-28d; in extreme cases 40-60d (but excisedembryos 2-4d only)

5-14d; inextreme cases21-40d

KNO3 *0.2% *0.2% 0.2%

GA3 *100-400 ppm 5 ppm 5-10 ppm

pre-chill 60d at 2°-5°Cwith high oxygenconc.

SUCCESSFULGERMINATION

TEST

light insensitive *insensitive orsensitive(promotory)

*insensitive or sensitive (promotory) insensitive orsensitive(promotory)

REGIMES(*most widely

applied treatments)

seed coat *file; chip (clip);excise embryo

excise embryo *excise embryo; pierce, remove over radicle *excise embryofrom endospermand seed coat;chip (clip)radicle end

scarification *mechanical; concH2SO4 for 15 min to4 h; absolute ethylalcohol for 20-72 h;percussion (shake)

pre-soak water at 50°-90°C for30 sec-24h

400 ppm GA3 or warm water for 24h *in water or 5%chlorox or leach(even withexcisedembryos) awayinhibitors withwater

after-ripen dry at 95°C for 6mins; dry over CaCI2

2-3m dry storage

PLANT FAMILIES

ANACARDIACEAE,CONVOLVULACEAE,GERANIACEAE,LEGUMINOSAE,

BALSAMINACEAE,CRUCIFERAE,LABIATAE,LINACEAE,

ACANTHACEAE, APOCYNACEAE,BALSAMINACEAE, BORAGINACEAE,CISTACEAE, DIPSACEAE,EUPHORBIACEAE, HYDROPHYLLACEAE,

COMPOSITAE(only)


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WITH SPECIESEXHIBITING THESECHARACTERISTICS

MALVACEAE,RHAMNACEAE,SAPINDACEAE

PLANTAGINACEAE,VIOLACEAE

LABIATAE, LIMNANTHACEAE,LOASACEAE, ONAGRACEAE,PASSIFLORACEAE, PLUMBAGINACEAE,POLEMONIACEAE, ROSACEAE,VALERIANACEAE, VERBENACEAE,ZYGOPHYLLACEAE

II. Seeds with only residual or no endosperm (NON-ENDOSPERMIC SEEDS) and mature independentembryos (that is embryos ready to germinate)

A. HARD SEED COAT

Hard (impermeable) seed coats are present which limit the entry of water for imbibition. Treatment for allowing water entry isnecessary and may be mechanical scarification, acid treatment, percussion, high temperature treatment or soaking. (SeeChapter 7, Volume I, for more information on treatments to overcome hardseededness.)

B. THIN SEED COAT WITH MUCILAGINOUS LAYER

Mucilaginous seed coats limit oxygen availability to the embryo after imbibition. Gibberellic acid is the most effective additivebut light and potassium nitrate are also helpful, particularly when treatment with the two agents is combined.

C. WOODY SEED COAT WITH INNER SEMI-PERMEABLE LAYER

Woody-textured and membranous multi-layered seed coverings cause blocks to germination which are most difficult toovercome. They readily admit water for imbibition but contain strong inhibitors which do not leach readily. Some of these areprobably located in the thin residual endosperm surrounding the embryo or within the cotyledons of the embryo and areblocked from leaching by the semi-permeable membranous testa incorporated in the coverings. Excised embryos germinatepromptly.

D. FIBROUS SEED COAT WITH SEPARATE SEMI-PERMEABLE MEMBRANOUS COAT

The Compositae family forms a specialised group which is similar to the preceding section. Embryos contain inhibitors in theircotyledons. Testa and thin endosperm form a separate membranous semi-permeable coat. An outer fibrous coat offers variousdispersal forms. Extracted embryos grow promptly if leaching is complete.

Summaries of seed anatomy, blocks to germination, successful germination test regimes and families exhibiting thesecharacteristics are provided in Table 17.2 for non-endospermic seeds.

Atwater has developed her ideas and experience almost to the stage of providing species prescriptions. Much of thisinformation is summarised in subsequent chapters, but the problems inherent in species prescriptions are discussed below.Note also that certain plant families have species in more than one category of seed anatomy (e.g. Solanaceae, see Tables17.1 and 17.2). If Tables 17.1 and 17.2 are used to develop germination test environments and dormancy-breakingtreatments, it should be remembered that combinations of different dormancy-breaking agents are more likely to be successfulthan treatment with a single agent alone.

Further reading

Atwater, B.R. (1978). Dealing with stop-go germination in flower seeds. Acta Horticulturae, 83, 175-179.

Atwater, B.R. (1980). Germination, dormancy and morphology of the seeds of herbaceous ornamental plants. Seed Scienceand Technology, 8, 523-573.

Mullet, J.H. (1981). Germinating those problem seeds. Australian Horticulture, December 1981, 61-67.

PRESCRIPTIONS FOR GERMINATING ALL ACCESSIONS OF A GIVEN SPECIES

This approach to determining germination test environments is exemplified by the Rules of the International Seed TestingAssociation (ISTA) and the Association of Seed Analysts of North America (AOSA). Commercial seed testing has evolved overthe past 130 years to provide a certain degree of assurance to farmers, growers and foresters that any seeds of agricultural,horticultural or arboreal crops that they may purchase are capable of germinating under normal field conditions, emergingabove the soil surface and developing into plants which yield an adequate crop. Thus, by assessing the quality of seed lotsbefore they are sown, commercial seed testing minimises the risk to the farmer, grower or forester of sowing seed lots whichdo not have the ability to produce an abundant crop. Since this assessment will be done by only one of many laboratories andsince there is also considerable international trade in seed of many species, assessments by different seed testing laboratoriesmust give similar results.

Compatibility between seed testing stations

To achieve compatibility of germination test results between different laboratories ISTA and AOSA co-ordinate and produceagreements between seed testing laboratories on the procedures to be applied for each species. We have discussed inChapters 4 and 5 (Volume I) how the proportion of seeds which will germinate in a germination test can be affected by the testenvironment. Thus if seed testing laboratories used very different germination test conditions the results obtained in differentlaboratories could differ substantially. To aid compatibility ISTA and AOSA seek agreement between laboratories as to themost appropriate environment for testing the germination of a given species. In theory this implies agreement upon a singlegermination test environment. In practice alternative environments are often specified for a given species. This is eitherbecause of disagreement between laboratories as to the single most appropriate environment, or because of the need toprovide alternative procedures for certain seed lots, or because of practical difficulties in providing certain environments (for


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example, alternating temperatures) in some laboratories.

The rules of the ISTA and AOSA list the prescribed germination test conditions for each species. These prescriptions specifythe substrata, temperatures and test duration which seed testing laboratories must adhere to, Additional directions areprovided for special treatments which may be required for certain species together with recommendations for breakingdormancy. These prescriptions and directions are summarised in the chapters which follow.

Now, these rules and directions are intended to give the most regular, rapid and complete germination for the majority ofsamples of seed of a particular species. To that end we presume the rules are successful, but how closely does the above aimcoincide with the problems facing those testing seeds within gene banks?

Prescriptions are not available for all species

First, ISTA and AOSA tend to only provide rules for those species in which certian individual seed testing stations haveknowledge of the germination behaviour of a large number of seed samples. Thus the species listed in the ISTA and AOSArules are those in which there is considerable trade in the sees; the seed-propagated crops. In contrast gene banks will oftenneed to germinate seeds of species which are not yet in substantial trade in the developed world (e.g. the tropical pasturespecies), true seeds of vegetatively propagated crops (e.g. potato, Solanum tuberosum), and seeds of species which do nothave crop status such as wild and weedy relatives of modern crop species (e.g. teosinte, Euchlaena mexicana). Thus the firstdrawback for gene banks of the ISTA/AOSA rules is that for many species on which gene banks require advice on germinationtest conditions no information is provided.

Prescriptions developed for modern cultivars

Secondly, within those species for which ISTA and AOSA prescribe test conditions the range of seed lots which seed testingstations test for germination, and upon the results of which ISTA precriptions are based, are generally limited to relatively non-dormant, high quality, modern, genetically homogeneous cultivars. That is in most cases the cultivars with which the ruleshave been developed are likely to have been subjected to considerable selection pressures. In contrast gene banks will betesting seeds of primitive cultivars and genetically heterogeneous landraces which may also show considerable dormancyand/or be of poor quality. Thus the second drawback for gene banks of the ISTA/AOSA rules is that the prescribed conditionsfor a particula species will not necessarily be suitable for those acessions within gene banks which are dissimilar to moderncultivars - or of poor quality.

Relative priorities of the aims of seed testing

Thirdly, for some crops seed testing stations may operate under considerable pressure at harvest time. For example, intemperate countries autumn sown cereal seeds are sown only five or six weeks after harvest. Thus seed testers may need tocomplete germination tests in as short a time as possible. Hence seed testing stations' aim of providing conditions which giverapid germination. Now in at lest one group of species - the temperate cereals - although the conditions prescribed by the rulesmay give rapid germination this is only achieved in our experience by using supra-optimal temperatures at the cost of theparallel aims of achieving regular and complete germination which are better achieved at much lower temperatures. In contrastto seed testing stations, gene banks are not under any pressure to give priority to rapid germination over achieving regular andcomplete germination. Thus the third drawback for gene banks of the ISTA/AOSA rules is that the aims of rapid and completegermination may not always be compatible. Note that this criticism does not apply to the rules for all crops. In the case of treespecies, for example, considerable periods are allowed for dormancy-breaking and germination test treatments by ISTA andAOSA rules, and the requirement for complete germination takes precedence over the requirement for rapid germination.

Field planting value

The purpose of commercial testing is to provide information on the comparative field planting value of different seed lots. Ingeneral the ISTA and AOSA prescriptions make no attempt to enable those viable seeds which will not germinate under fieldconditions to germinate in the laboratory test. If, for example, dormancy is a problem in laboratory tests, but not in field sowings(for example, due to differences in temperature between the two regimes) then directions to break dormancy must be providedin the rules - otherwise the field planting value may be underestimated. However, where dormancy is a problem in bothlaboratory tests and field sowings then to obtain an indication of field planting value the rules may not necessarily providedormancy-breaking directions - since the removal of dormancy in laboratory tests may result in an overestimate of field plantingvalue.

Again whether or not this objective of ISTA or AOSA prescriptions and recommendations makes them unsuitable for viabilitytesting in gene banks depends on the species. For example, in tree species directions for dormancy removal are suggested toenable viability to be estimated in laboratory tests and these directions must also be followed in field sowings if laboratoryresults are to provide a reasonable estimate of field planting value. In contrast in some species where hardseedednessprevents germination no recommendation is made to render the seed coat permeable. Instead the proportion of hard seeds isreported. Thus the fourth drawback of ISTA/AOSA rules is that no attempt may be made to overcome a wide range ofphysiological phenomena which may prevent the germination of viable seeds under both laboratory and field conditions. Ofthese phenomena the most important are hardseededness and strong dormancy. In contrast to seed testing stations, genebanks aim to provide conditions which will enable all viable seeds to germinate both in laboratory tests and in subsequent fieldor glasshouse sowings.

Other sources of prescriptions

Numerous groups of workers have attempted to provide prescriptions for germination tests of species for which no ISTA/AOSArules are available or which overcome certain of the deficiencies of the ISTA and AOSA prescriptions for species where rulesare available. Particular emphasis has been placed on overcoming dormancy since this can be an important problem for plantbreeders, and also for seed testers if considerable dormancy is experienced when the seeds are tested but which is likely to belost during storage before sowing.


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However, much of this information is also of limited use in gene banks. This is because the majority of such information relatesto the application of single dormancy breaking agents alone. In Chapter 5 (Volume I) it was explained that if this approach isused then the treatment concentration (if a chemical agent) or duration (if another form of treatment) required to breakdormancy in the most dormant seeds would be likely to damage less dormant seeds, particularly if these are also of poorquality.

The aim of developing a species prescription for use in gene banks

We believe that the ultimate desirable aim of a prescriptive test for a given species is to be able to provide within one testenvironment or procedure those conditions which enable all viable seeds of that species - whether strongly dormant,moderately dormant, weakly dormant or non-dormant, and of high or low quality - to be able to germinate. This means thatany stimulus or combination of stimuli used to promote the germination of dormant seeds must not be damaging to other non-dormant seeds in the sample or to intolerant genotypes.

Developing a prescription for a species

Whether this aim can in fact always be achieved is a matter for debate, but two considerations are important in attempting toachieve it. The first is to ensure that the germination test temperature is suitable for poor quality seeds; within this rangeattempts can then be made to determine the most suitable temperature regime which will minimise the expression ofdormancy. In certain species this first step may result in an adequate single germination test environment without any need forfurther treatment. In temperate cereals this appears to be the case, where a single low constant temperature is appropriate forboth purposes. However, in other species simple solutions of this type may not be available and so it is necessary to take asecond step and provide further stimuli to break dormancy which do not impair the germination of less dormant seeds. In ouropinion this is often best attempted by combining more than one additional stimulus with all stimuli being applied at relativelylow dose-levels. This is an attempt to utilise the positive interactions observed between many dormancy-breaking agents (seeChapter 5, Volume I). The treatments developed for Oryza spp. and Vitis spp. provide examples of this type of procedure(these and other prescriptions are described in the appropriate chapters of this volume).

Gene banks considering developing their own germination test prescriptions may be discouraged by the wide range of differenttreatments - and the even wider range of different treatment combinations - which may be of potential benefit in promoting thegermination of viable seeds. Consequently it is worthwhile considering which factors should receive priority in anyinvestigation. We believe that the three most important factors to consider first are to ensure that the seeds have imbibedmoisture without damage (see Chapter 7, Volume I) and then to attempt to determine the most suitable temperature (probablyalternating temperature, see Chapter 5, Volume I) and light (see Chapter 6, Volume I) regimes for germination.

Provided a prescription is already available for a species which is suitable for application in a gene bank then this would be thesimplest approach for gene banks to use. However, even where a prescription is well-tried, gene banks will still need to bealert to the possibility that some accessions may be discovered for which the prescription does not appear to work.Furthermore, for many years to come, there will continue to be a large number of species for which there are no adequateprescriptions. Accordingly alternative approaches to providing suitable germination test environments are also considered inthis handbook.

Use of tetrazolium test to determine efficacy of a prescription

Although a gene bank can never be absolutely certain about the efficacy of any approach to germinating seeds, do not forgetthat the efficacy of a species prescription on a particular accession can be tested by comparing the results of the germinationtest with that of the topographical tetrazolium test (see Chapter 11, Volume I) on a sub-sample of the seed lot and by alsoapplying a tetrazolium test to the seeds remaining ungerminated at the end of the test. In this way one can determine theproportion (if any) of viable seeds which are either killed by the germination test procedure or remain dormant at the end of thegermination test procedure.

In the above we have detailed objections to the wholesale and unquestioning adoption of either ISTA or AOSA prescribedgermination test procedures as gene bank germination tests. However, this certainly does not mean that such prescriptionsshould be ignored by gene banks. The ISTA and AOSA rules summarise a wealth and depth of expertise and experiencewhich is unmatched. Thus where available, the prescribed conditions represent a control against which any proposedalternative should be tested; and, despite our earlier comments, certain of these prescriptions will be suitable for gene bankuse.

TAXONOMIC ALGORITHMS TO DETERMINE APPROPRIATE GERMINATION TEST ENVIRONMENTS

As mentioned above, germination test prescriptions are available for only a small proportion of seed-producing species. Whatenvironment should be chosen to test the germination of seed accessions of species for which no prescription or noacceptable prescription is available? This is exactly the problem which faced staff at the Royal Botanic Gardens Kew,Wakehurst Place Gene Bank, for almost all their accessions, because this gene bank deals entirely with seed collections ofspecies which do not have great commercial significance. The method that they developed to answer the question posedabove provides another approach to developing appropriate germination test regimes for seed accessions maintained in genebanks.

Algorithm: the shortest series of tests to define a suitable test environment

This approach acknowledges that the most suitable environment for testing each accession is unknown. Thus, instead of asingle prescription, an attempt has been made to develop the shortest series of tests which is likely to give a solution to theproblem of defining a suitable test environment. The decision as to which test to try next depends on the results obtained in theprevious test(s), and so we call this series of tests an algorithm. For gene banks a truly universal algorithm would be oneappropriate to all seed-producing species, irrespective of taxonomy. However, such an approach would be rather unwieldy and


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require a large number of tests. Certain similarities in the response of seed germination to environment for species within afamily can be recognised. Consequently this approach provides a separate algorithm for each family where sufficientknowledge has been accumulated.

Where available, the algorithms developed by the staff at the Wakehurst Place Gene Bank are presented according to family inChapters 18 to 75. However, it is necessary here to provide some explanation of how the algorithms were developed, theirpossible faults, and how they should be applied.

The development of the RBG Kew Wakehurst Place algorithms

Storage at the Wakehurst Place Gene Bank began in 1974. Since then all accessions entered into storage have been testedfor germination as soon as possible thereafter. In many cases accessions were tested in more than one germination testenvironment, the reason for this being an inadequate proportion (less than 85%) of seeds germinating in the first test. Thus formany accessions it was possible to compare germination test results over a number of different environments with and withoutthe imposition of various additional dormancy-breaking treatments. For each accession the best germination test regime wasnoted, and this regime was then used for all subsequent germination tests of that accession. In addition the results of all thedifferent germination tests carried out on each accession were filed according to species to provide a growing information bankon the response of seed germination to different test conditions. Table 17.3 provides an example of the information generatedand filed from such tests for a single accession of Veronica verna.

TABLE 17.3 Example of germination test results recorded by staff at the Wakehurst Place Gene Bank used in subsequentanalyses to determine algorithms for determining suitable test regimes: germination (%) and classification of non-germinatingseeds after testing seeds of a 1977 collection of Veronica verna under four different regimes (100 seeds per test).

Test Date Germination Test regime Total Test Duration(days)

Cumulative Germination(%)

at end of test

Non-germinated seeds at end of test1

Fresh Mouldy Empty

16/12/77 21°C 42 0 100 0 0

16/12/77 21°/11°C (12h/12h) 157 48 52 0 0

18/4/78 26°C for 28 days(imbibed) then 11°C

48 100 0 0 0

18/4/78 2°C 73 88 12 0 0

1 expressed as the percentage of the original number of seeds tested

The two original tests on seeds from this accession, begun on 16/12/77, demonstrated a high proportion of dormant seeds(those designated as fresh at the end of the test - see Chapters 10 and 11, Volume I). At 21°C all seeds exhibited dormancy(Table 17.3). The alternating temperature 'regime of 21°/11°C was only partly successful in breaking this dormancy (Table17.3). Therefore towards the end of the latter test, a decision was made to sample more seeds from the accession and begintwo additional germination tests in different conditions (which are known to promote germination in several species). One ofthese additional regimes - consisting of a preliminary period during which the imbibed seeds were exposed to 26°C(sometimes described as a warm stratification treatment) followed by subsequent exposure to 11°C - was entirely successful atbreaking dormancy, enabling all the seeds tested to germinate (Table 17.3). Consequently this regime will be used for anysubsequent germination tests to monitor the viability of this accession.

Over the past ten years or so a considerable amount of information on the response of seed germination to various testregimes (similar to that described above for Veronica verna) has been generated by staff at Wakehurst Place. This rawinformation has subsequently been analysed, principally by Mr. S. Linington, in the following manner. For a particular family allthe routine test data for all accessions (each similar in form to that provided in Table 17.3) were examined. It was (arbitrarily)decided that test regimes in which 85%, or more, of the full seeds germinated would be classified as successful. For eachaccession all the successful regime(s) (if there were any) were listed. For example, two regimes would be listed as successfulfor the accession shown in Table 17.3: 2°C constant; and 28 days at 26°C followed by 11°C constant.

When this first stage of analysis was completed a pattern of successful regimes within each family was sought. Emphasis wasplaced on determining those simpler regimes (that is those employing few dormancy-breaking agents) which were successfulfor the greatest number of accessions. These regimes are those which are now used as the first step in the algorithms whichhave been developed. More complicated regimes (involving the use of many dormancy-breaking treatments) which aresuccessful for fewer accessions are then provided as subsequent alternatives (later steps) in the algorithm if the first step doesnot provide a suitable environment for germination testing.

Possible limitations of the algorithms

There are a number of possible faults inherent in this method of analysis. First, by excluding from the analysis all accessionswhich failed to reach 85% germination under any test regime, the response of accessions which are either very dormant, orvery poor quality and vulnerable in certain germination test environments may have been ignored. Secondly, it is based ontests of accessions within the Wakehurst bank which, at least in the past, were biased towards collecting in temperate regions.Not only may this result in an algorithm more suited to temperate collections, but it may also result in algorithms more suited tocertain tribes within each family. For example, the algorithm for the Leguminosae was based largely on accessions of thePapilionoideae tribe and little data was available for accessions from either the Caesalpinoideae or Mimosoideae tribes. Finallyit should be noted that the analysis was based on data obtained over only a limited range of treatments. Other, untested,treatments may be superior and there is as yet no way of knowing whether this will be the case.

Success of the algorithms may vary between families

Despite these reservations, the algorithm approach to determining germination test environment works well at Wakehurst and


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is likely to be useful in other gene banks. Tests at Wakehurst on previously untested accessions using the algorithms for theAmaranthaceae, Gramineae, Leguminosae and Solanaceae families (provided in Chapters 20, 39, 43 and 67 respectively)have shown that 100%, 50%, 90% and 75% of accessions within each family reached 85% germination or greater. In view ofthe importance of the Gramineae in food production the limited success of the algorithm for this family is disappointing. Onegroup of species within the Gramineae whose seed is particularly difficult to germinate is the tropical pasture species.Unfortunately tests on five seed lots of tropical pasture species at Reading (Table 17.4) confirm that the present algorithm forthe Gramineae (Chapter 39) is not particularly helpful for determining germination test environment in these species.

Demonstration of the use of an algorithm

Nevertheless, Table 17.4 can be used to explain the method of applying the algorithm. Table 17.4 should be studied togetherwith the Gramineae algorithm provided in Chapter 39. In Table 17.4 every feasible combination of dormancy-breaking agentswithin the algorithm was applied to the seeds, amounting to some 24 separate test environments. Note, however, that only afew of these tests would have been applied if the algorithm had been followed. The pathway through the tests which wouldhave been followed if the Gramineae algorithm had been applied is shown by the boxed values in Table 17.4, the sequencebeing from the top of the table to the bottom.

Table 17.4 Normal germination (expressed as percentage of full seeds tested) after 28 days in germination test [lowerfigures in square brackets after 56 days] for five tropical grass seed lots. If the Gramineae algorithm (Chapter 39) had beenfollowed in the manner intended only the tests in square boxes would have been investigated (see text).

Treatment

SPECIES

Brachiaria decumbens Brachiaria humidicola (Gatton Panic)Panicum maximum

(Green Panic)Panicum maximum

(Riversdale Guinea)Panicum maximum

Temperature, °C Temperature, °C Temperature, °C Temperature, °C Temperature, °C

Control(constant

temperature)

20 25 35/20 (15/30,10/30) 20 25 35/20 (15/30,10/30) 20 25 35/20 (15/30,10/30) 20 25 35/20 (15/30,10/30) 20 25 35/20 (15/30,10/30)

[4] [1] [0] [4] [39] [25] [18] [18] [2] [0][5] [3] [0] [4] [39] [26] [18] [18] [3] [0]

Alternatingtemperature

[19] (10,10) [21] (74,73) [46] (90,92) [18] (13,7) [31] (57,69)[22] ([10],[15]) [57] ([75],[77]) [49] ([92],[93]) [18] ([15],[8]) [44] ([58],[70])

10-3 MKNO3

3 3 [28] (13,17) 0 1 [18] (66,-) 61 31 [59] (93,82) 14 14 [13] (14,14) 3 0 [40] (64,79)[4] [3] [30] ([13],[19]) [1] [2] [64] ([67],-) [61] [31] [64] ([97],[90]) [16] [14] [13] ([17],[14]) [6] [0] [51] ([64],[80])

Dehusk

15 14 65 0 2 [14] 11 8 7 14 15 [18] 1 2 35[17] [15] [66] [0] [2] [45] [12] [10] [12] [14] [15] [19] [10] [2] [41]

Dehusk +KNO3

14 15 [63] 4 3 12 17 11 [9] 14 21 13 18 3 [61][15] [16] [63] [4] [4] [40] [19] [13] [26] [14] [22] [14] [19] [3] [69]

8 week pre-chill

0 0 0 0 0 [3] 33 21 16 0 0 [2] 35 54 59[0] [0] [0] [0] [0] [4] [34] [21] [18] [0] [0] [2] [35] [54] [62]

Dehusk +pre-chill

1 7 38 0 0 5 11 27 25 0 2 2 35 31 46

[2] [7] [38] [1] [0] [12] [11] [27] [26] [0] [3] [4] [38] [31] [49]

KNO3 +pre-chill

1 1 1 1 0 4 8 20 [25] 2 2 2 49 62 59[1] [1] [1] [1] [0] [5] [8] [20] [25] [2] [2] [2] [49] [62] [62]

Dehusk +KNO3 +pre-chill

0 9 [35] 0 0 6 24 18 30 4 2 7 31 45 [62][0] [10] [35] [2] [0] [9] [24] [18] [30] [5] [3] [9] [33] [45] [62]

Viability(tetrazolium

test)

[94] [75] [97] [25] [76]

Three details of the work reported in Table 17.4 differ from the Gramineae algorithm provided by the Wakehurst Place GeneBank. First the germination test temperatures are not identical, but are within 1°C - except for the upper limit of the alternatingtemperature regime where the difference is 2°C. These minor differences are unimportant and result from differences in thestandard temperatures available at Reading and Wakehurst. Secondly the comment in the algorithm to test at constanttemperatures either below 20°C or above 25°C depending upon which gave the higher test result was not followed, sinceprevious experience with dormant seed lots of these species indicated that alternating temperatures were essential to promotegermination. Thirdly, following on from the last point, seeds were also tested for germination at the two additional alternatingtemperature regimes of 15°/30°C and 10°/30°C.

To enable the reader to understand fully the use of the algorithm, an explanation is provided below of the sequence of testsand decisions that would have been applied for tests on the accession of Brachiaria decumbens. The first stage in thealgorithm was to test the seeds at constant temperatures of 20°C and 25°C. These test results were inadequate (less than85% germination).


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Now, according to the algorithm (Chapter 39) the next step would have been to look for trends in the results of these two tests(i.e. was germination greater at the higher or lower temperature?) and test for germination at either a more extreme higher or amore extreme lower constant temperature accordingly. This, however, was not done for the reasons given above and weproceeded to the second step of the algorithm which was to test a fresh sample of the seeds in an alternating temperatureregime of 35°/20°C (the nearest regime available to the standard 33°/19°C Wakehurst regime). The germination obtained inthis test was substantially greater than at either of the two constant temperatures, but again inadequate as a germination testregime. Consequently further tests were required.

The third step according to the algorithm was to test a fresh sample of the seeds in a medium containing 10-3 M KNO3 at, inview of the difference observed between the results at steps 1 and 2, an alternating temperature of 35°/20°C. Again the resultof this test at step 3 was an improvement on the result at step 2 but inadequate as a germination test regime since 85%germination was not achieved.

Thus the fourth step was to remove the seed covering structures from a fresh sample of the seeds and test in a mediumcontaining 10-3 M KNO3 (since the result at step 3 was greater than the result at step 2) at an alternating temperature of35°/20°C. Once again the result of the test at this step was an improvement on the previous test result (suggesting thatdehusking was a worthwhile treatment), but the percentage of seeds germinating was still unsatisfactory.

Consequently the final step in the algorithm was applied. Namely dehusked imbibed seeds were pre-chilled at 3°-5°C for 8weeks and then transferred to an alternating temperature regime of 35°/20°C in a medium containing 10-3 M KNO3.Unfortunately this test resulted in substantially fewer seeds germinating than the previous test. Thus the conclusion reached byusing the algorithm is that dehusked seeds of this accession of Brachiaria decumbens should be tested on a substratecontaining 10-3 M KNO3 in an alternating temperature regime of 35°/20°C.

Note that the decision making procedure at each step in the algorithm did not result in the algorithm missing more favourabletreatment combinations. For example, if the algorithm had been followed dehusked seeds would not have been tested at35°/20°C without 10-3 M KNO3 in the germination medium. In fact this treatment gave a very similar result (that is there was no

significant difference) to the treatment with dehusked seeds which did include 10-3 M KNO3 in the germination medium. Thus it

appears that the use of 10-3 M KNO3 in the medium is in fact not necessary, but its use (apparently necessary if the algorithmhad been followed) does not result in a significantly lower proportion of seeds germinating. In that sense we can regard thedecision-making procedure within the algorithm as being successful, but unfortunately the algorithm itself was not successfulsince the level of viability indicated by the tetrazolium test was not achieved in these germination tests.

Sensitivity of germination to the temperature regime

In addition to all the possible germination test regimes according to the Gramineae algorithm, Table 17.4 also presents resultsof tests carried out in two additional alternating temperature environments (15°/30°C and 10°/30°C) and also in combinationwith 10-3 M KNO3 (where sufficient seeds were available). These results suggest that, for some seed lots at least, those genebanks testing tropical grass species would be well advised to include additional alternating temperature regimes as the secondstep of the algorithm. For three of the seed lots tested here small differences in the alternating temperature regime greatlyinfluenced the proportion of seeds which germinated (Table 17.4). Not only did this result in almost full germination of viableseeds, but it would have avoided the need for several stages of the algorithm in the case of Gatton Panic. (See the section onthe genus Panicum, Chapter 39, for advice on an appropriate alternating temperature regime for this species.)

In passing note that the results shown in Tables 17.3 and 17.4 emphasise that seed germination can be particularly sensitiveto the temperature regime in which the seeds are tested. It cannot be overemphasised that gene bank staff must makestrenuous efforts to get this right first before embarking on investigations as to the efficacy of other dormancy-breaking agents.

Possible use of ungerminated seeds from prior step i

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